title: Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities.
abstract: Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.
authors.affiliation: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Belli
authors.forename: Priska M
authors.initials: PM
authors.lastname: Elsener
authors.forename: Heidi
authors.initials: H
authors.lastname: Wunderli-Allenspach
authors.forename: Stefanie D
authors.initials: SD
authors.lastname: Krämer
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D016880:Anisotropy; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000502:physiology*; D004790:Enzyme Induction / Q000187:drug effects; D005079:Excipients; D008051:Lipid Bilayers; D008081:Liposomes / Q000737:chemistry; D008567:Membranes, Artificial; D051379:Mice; D011412:Propanolamines / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000316:Adrenergic alpha-Agonists; D000972:Antineoplastic Agents, Phytogenic; D002121:Calcium Channel Blockers; D005079:Excipients; D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D011412:Propanolamines; D015060:1,2-Dipalmitoylphosphatidylcholine; C011550:talinolol; D014747:Vinblastine; D002784:Cholesterol; D014700:Verapamil; D000251:Adenosine Triphosphatases; D024502:alpha-Tocopherol